The readout of genome information is controlled by transcriptional regulatory elements, but a comprehensive view of the combinatorial control by these DNA sequences, which bind regulatory protein and/or the modified histones in regulating gene transcription, is clearly preliminary. We propose an experimental strategy for comprehensive determination of such functional elements in human DNA. This strategy is based on our extensive knowledge of the transcriptional regulatory proteins, and the demonstrated utility of a method that we previously developed to identify genomic binding sites for DNA-binding proteins in living cells. The method, known as genome-wide location analysis or ChiP-on-Chip, involves immunoprecipitation of the DNA associated with a particular protein from crosslinked cells, followed by quantitative amplification and simultaneous detection of the enriched sequences with DNA microarray technologies. Our experimental strategy to map transcriptional regulatory elements involves the application of genome-wide location analysis to a panel of well-characterized regulatory proteins and histones with specific modifications, known to generally associate with transcriptional regulatory elements in vivo. Identification of their genomic binding sites will allow us to determine the sequence features in the human genome that carry out transcriptional regulatory function. To demonstrate the feasibility of this strategy to the genomic sequences specified by the ENCODE project, we will first design and produce DNA microarrays to represent all the non-repetitive sequences in these genomic regions to map three types of transcriptional regulatory elements in three model cell types. We will identify gene promoters by mapping the genomic sequences associated with RNA polymerase II and the general transcription factor TFIID in cells, and identify enhancer elements by mapping the genomic sequences associated with transcriptional co-activators, acetylated histones H3 and H4 in cells, and identify the repressed and/or silenced DNA by mapping the genomic sequences associated with the heterochromatin binding protein HP1, tri-methylated histone H3 (lysine 9), and transcriptional co-repressors in cells. We will integrate results from our studies with genome-wide expression profiles, comparative genomics analyses and external data sets to gain a comprehensive view of the transcriptional regulatory elements in human DNA. We expect to elucidate the general principles that govern the genomic distribution of transcriptional regulatory elements, and understand the molecular mechanisms that control genome expression in human cells.